Entry and Release of Hepatitis C Virus in Polarized Human Hepatocytes

Abstract

Hepatitis C virus (HCV) infects primarily hepatocytes that are highly polarized cells. The relevance of cell polarity in the HCV life cycle has only been addressed in distant models and remains poorly understood. Although polarized epithelial cells have a rather simple morphology with a basolateral and an apical domain, hepatocytes exhibit complex polarization structures. However, it has been reported that some selected polarized HepG2 cell clones can exhibit a honeycomb pattern of distribution of the tight-junction proteins, typical of columnar polarized epithelia which can be used as a simple model to study the role of cell polarization in viral infection of hepatocytes. To obtain similar clones, HepG2 cells expressing CD81 (HepG2-CD81) were used and clones were isolated by limiting dilutions. Two clones exhibiting a simple columnar polarization capacity when grown on semi-permeable support were isolated and characterized. To test the polarity of HCV entry and release, our polarized HepG2-CD81 clones were infected with cell culture derived HCV. Our data indicate that HCV binds equally to both sides of the cells, but productive infection occurs mainly when the virus is added at the basolateral domain. Furthermore, we also observed that HCV virions are released from the basolateral domain of the cells. Finally, when polarized cells were treated with oleic acid and U0126, a MEK inhibitor, to promote lipoprotein secretion, a higher proportion of infectious viral particles of lower density was secreted. This cell culture system provides an excellent model to investigate the influence of cell polarization on the HCV life cycle.IMPORTANCE Hepatitis C is a major health burden with approximately 170 million persons infected worldwide. Hepatitis C virus (HCV) infects primarily hepatocytes that are highly polarized cells with a complex organization. The relevance of cell polarity in the HCV life cycle has been addressed in distant models and remains unclear. Hepatocyte organization is complex with multiple apical and basolateral surfaces. A simple culture model of HepG2 cells expressing CD81 that are able to polarize with a unique apical and basolateral domain was developed to study HCV infection. With this model, we demonstrated that HCV enters and exits hepatocytes by the basolateral domain. Furthermore, lower density viral particles were produced in conditions that promote lipoprotein secretion. This cell culture system provides a useful model to study the influence of cell polarization on HCV infection.

Role of the cytosolic domain of occludin in trafficking and hepatitis C virus infection

“…This result suggests that the localization of OCLN to the TJs is not an absolute prerequisite for infection. In line with this hypothesis, a fraction of the CLDN1 pool was found to be expressed together with CD81 and SR‐BI at the basolateral membrane of clone 1sc3‐polarized cells . Moreover, in this model, HCV entry has been shown to occur preferentially from the basolateral pole of the cell.…”

“…In order to further characterize the subcellular localization of OCLN‐C18 mutant, we co‐transduced OCLN‐KO 1sc3 cells with lentiviral expression vectors for OCLN mutants and with lentiviral expression vectors for CD81 or CLDN1 fused to a fluorescent protein (green fluorescent protein and cerulean, respectively). Whereas CLDN1 localizes at TJ, CD81 has been shown to be only present at the basolateral pole of 1sc3 cells . In agreement with the data obtained in ZO‐1 co‐labeling assay, wild‐type OCLN colocalized with CLDN1 in the areas of cell‐to‐cell contact while OCLN‐C15 accumulated in the cytosol.…”

“…However, the deletion of the C‐terminal domain might potentially affect its ability to associate with TJs and modulate their functions. In order to determine the functionality of the different OCLN mutants in a more physiologically relevant system, we used polarized Huh‐7‐derived cells . Thus, Huh‐7 clone 1sc3 cells that polarize in the presence of DMSO were transduced by lentiviral vectors expressing wild‐type or mutants OCLN.…”

“…HEK293T/17 cells (ATCC), OCLN‐knockout human hepatic Hu7.5.1‐8‐derived OKH4 cells, Huh‐7.5, Huh‐7, Huh‐7 clone 1sc3 cells were cultured in Dulbecco's modified eagle medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum, 2 mM GlutaMax and nonessential amino acids (Life Technologies).…”

“…Noteworthy, CD81 and OCLN determine the tropism of HCV for human cells. Since CLDN1 and OCLN are TJ proteins, it was initially thought that HCV virions would migrate to TJs for internalization; however, experimental data could not support this hypothesis until recently . Indeed, imaging of HCV entry in a three‐dimensional polarized hepatoma system revealed a sequential engagement of the entry factors with an initial localization of HCV with SR‐BI and CD81 at the basolateral membrane and a subsequent accumulation of the virus at the TJs in an actin‐dependent manner …”